scholarly journals Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells

2002 ◽  
Vol 282 (6) ◽  
pp. C1361-C1373 ◽  
Author(s):  
Lucia Formigli ◽  
Fabio Francini ◽  
Elisabetta Meacci ◽  
Massimo Vassalli ◽  
Daniele Nosi ◽  
...  
Keyword(s):  
Specific Inhibitor ◽  
Cell Contraction ◽  
Cytoskeletal Rearrangement ◽  
Edg Receptors ◽  
Inositol 1,4,5 Trisphosphate ◽  
Confocal Fluorescence ◽  
Sphingosine 1 Phosphate ◽  
Intracellular Ca ◽  
Confocal Fluorescence Imaging ◽  
Differentiation Gene

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349–357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.

Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate

2001 ◽  
Vol 29 (6) ◽  
pp. 825-830 ◽  
Author(s):  
C. Pilquil ◽  
Z.-C. Ling ◽  
I. Singh ◽  
K. Buri ◽  
Q.-X. Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Activation ◽  
Growth Factor Receptors ◽  
Exogenous Lipid ◽  
Edg Receptors ◽  
Sphingosine 1 Phosphate ◽  
Lipid Phosphate ◽  
Lipid Phosphate Phosphatases ◽  
Differentiation Gene

The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for ‘ecto-phosphatase’ activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.

cGMP abolishes agonist-induced [Ca2+]ioscillations in human bladder epithelial cells

2001 ◽  
Vol 281 (6) ◽  
pp. F1067-F1074 ◽  
Author(s):  
H. Y. Kwan ◽  
Y. Huang ◽  
S. K. Kong ◽  
X. Yao
Keyword(s):  
Epithelial Cells ◽  
Prolonged Exposure ◽  
Specific Inhibitor ◽  
Cytosolic Calcium ◽  
Inhibitory Effect ◽  
Calcium Oscillations ◽  
Protein Kinase G ◽  
Human Bladder ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

First published August 9, 2001; 10.1152/ajprenal.00031.2001.—Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular Ca2+ concentration ([Ca2+]i ) while avoiding prolonged exposure to constantly elevated [Ca2+]i. In this study, we demonstrated that agonists could induce Ca2+oscillations in human bladder epithelial cells. Application of 10 μM acetylcholine or 200 nM bradykinin triggered an initial Ca2+ transient that was followed by periodic [Ca2+]i oscillations. The oscillations did not depend on extracellular Ca2+. 8-Bromoguanosine 3′,5′-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca2+]i oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to IP3 receptor-mediated Ca2+ release.

scholarly journals Inositol 1,4,5-Trisphosphate Signaling Regulates Rhythmic Contractile Activity of Myoepithelial Sheath Cells in Caenorhabditis elegans

2004 ◽  
Vol 15 (8) ◽  
pp. 3938-3949 ◽  
Author(s):  
Xiaoyan Yin ◽  
Nicholas J.D. Gower ◽  
Howard A. Baylis ◽  
Kevin Strange
Keyword(s):  
Caenorhabditis Elegans ◽  
Contractile Activity ◽  
Receptor Gene ◽  
Tyrosine Kinase Receptor ◽  
Sheath Cell ◽  
Cell Contraction ◽  
C Elegans ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Sheath Cells

Intercellular communication between germ cells and neighboring somatic cells is essential for reproduction. Caenorhabditis elegans oocytes are surrounded by and coupled via gap junctions to smooth muscle-like myoepithelial sheath cells. Rhythmic sheath cell contraction drives ovulation and is triggered by a factor secreted from oocytes undergoing meiotic maturation. We demonstrate for the first time that signaling through the epidermal growth factor-like ligand LIN-3 and the LET-23 tyrosine kinase receptor induces ovulatory contractions of sheath cells. Reduction-of-function mutations in the inositol 1,4,5-trisphosphate (IP3) receptor gene itr-1 and knockdown of itr-1 expression by RNA interference inhibit sheath contractile activity. itr-1 gain-of-function mutations increase the rate and force of basal contractions and induce tonic sheath contraction during ovulation. Sheath contractile activity is disrupted by RNAi of plc-3, one of six phospholipase C-encoding genes in the C. elegans genome. PLC-3 is a PLC-γ homolog and is expressed in contractile sheath cells of the proximal gonad. Maintenance of sheath contractile activity requires plasma membrane Ca2+ entry. We conclude that IP3 generated by LET-23 mediated activation of PLC-γ induces repetitive intracellular Ca2+ release that drives rhythmic sheath cell contraction. Calcium entry may function to trigger Ca2+ release via IP3 receptors and/or refill intracellular Ca2+ stores.

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors

2002 ◽  
Vol 362 (2) ◽  
pp. 349-357 ◽  
Author(s):  
Elisabetta MEACCI ◽  
Francesca CENCETTI ◽  
Lucia FORMIGLI ◽  
Roberta SQUECCO ◽  
Chiara DONATI ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Biological Effects ◽  
Cell Types ◽  
Confocal Laser Scanning Microscope ◽  
C2c12 Cells ◽  
Confocal Laser ◽  
C2c12 Myoblasts ◽  
Edg Receptors ◽  
Sphingosine 1 Phosphate ◽  
Differentiation Gene

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Confocal Fluorescence Imaging of Photosensitised DNA Denaturation in Cell Nuclei

2005 ◽  
Author(s):  
Tytus Bernas ◽  
Elikplimi K. Asem ◽  
J. Paul Robinson ◽  
Peter R. Cook ◽  
Jurek W. Dobrucki
Keyword(s):  
Fluorescence Imaging ◽  
Dna Denaturation ◽  
Cell Nuclei ◽  
Confocal Fluorescence ◽  
Confocal Fluorescence Imaging

scholarly journals Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum.

1994 ◽  
Vol 5 (1) ◽  
pp. 97-103 ◽  
Author(s):  
I Bezprozvanny ◽  
S Bezprozvannaya ◽  
B E Ehrlich
Keyword(s):  
Lipid Bilayers ◽  
Inhibitory Action ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Single Channels ◽  
Ca Channels ◽  
Open Time ◽  
Inositol 1,4,5 Trisphosphate ◽  
Insp3 Receptors ◽  
Intracellular Ca

Effects of the xanthine drug caffeine on inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels from canine cerebellum were studied using single channels incorporated into planar lipid bilayers. Caffeine, used widely as an agonist of ryanodine receptors, inhibited the activity of InsP3-gated Ca channels in a noncooperative fashion with half-inhibition at 1.64 mM caffeine. The frequency of channel openings was decreased more than threefold after addition of 5 mM caffeine; there was only a small effect on mean open time of the channels, and the single channel conductance was unchanged. Increased InsP3 concentration overcame the inhibitory action of caffeine, but caffeine did not reduce specific [3H]InsP3 binding to the receptor. The inhibitory action of caffeine on InsP3 receptors suggests that the action of caffeine on the intracellular Ca pool must be interpreted with caution when both ryanodine receptors and InsP3 receptors are present in the cell.

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

1999 ◽  
Vol 276 (5) ◽  
pp. C1115-C1120 ◽  
Author(s):  
Karl Dreja ◽  
Per Hellstrand
Keyword(s):  
Calcium Release ◽  
Cyclopiazonic Acid ◽  
Differential Modulation ◽  
Transient Responses ◽  
Inositol 1,4,5 Trisphosphate ◽  
Arterial Tissue ◽  
Intracellular Ca ◽  
Rat Tail

To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) ord- myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 μM), 5-HT (10 μM), or AVP (0.1 μM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 μM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]iand involving ryanodine- but not IP3 receptor-mediated Ca2+release.

scholarly journals Cross-talk between Native Plasmalemmal Na+/Ca2+Exchanger and Inositol 1,4,5-Trisphosphate-sensitive Ca2+Internal Store inXenopusOocytes

2004 ◽  
Vol 279 (50) ◽  
pp. 52414-52424 ◽  
Author(s):  
Luisa M. Solís-Garrido ◽  
Antonio J. Pintado ◽  
Eva Andrés-Mateos ◽  
María Figueroa ◽  
Carlos Matute ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Surface Membrane ◽  
Rabbit Serum ◽  
Caged Compounds ◽  
Reverse Transcription Pcr ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca ◽  
Functional Consequences ◽  
Extracellular Sodium ◽  
Cortical Endoplasmic Reticulum

Because the presence of a native plasmalemmal Na+/Ca2+exchange (NCX) activity inXenopus laevisoocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP3R) that controls intracellular Ca2+in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP3Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca2+mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+-activated chloride current, ICl(Ca), as a result of the Ca2+rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent “Ca2+extrusion” via NCX; and (v) blocking NCX enhanced the ICl(Ca)elicited by InsP3but not by Ca2+photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the “Ca2+influx” but not for the serum-elicited “Ca2+efflux” mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.

Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils

2000 ◽  
Vol 113 (15) ◽  
pp. 2737-2745
Author(s):  
S.I. Anderson ◽  
N.A. Hotchin ◽  
G.B. Nash
Keyword(s):  
Cytochalasin D ◽  
Calpain Inhibitor ◽  
Cytoskeletal Rearrangement ◽  
Activated Platelets ◽  
Binding Inhibition ◽  
Formyl Peptide ◽  
Intracellular Ca ◽  
Rapid Activation ◽  
Dose Dependent

When rolling adherent neutrophils are stimulated, they rapidly immobilize through activation of integrin CD11b/CD18, and then modulate attachment through this integrin to allow migration. We investigated links between cytoskeletal rearrangement and changes in function of integrin CD11b/CD18 in neutrophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin-polymerizing agent jasplakinolide became rolling adherent on monolayers of activated platelets, but could not use CD11b/CD18 to become immobilised when fMLP was perfused over them. If treated with jasplakinolide after fMLP, the cells stopped migrating but could not detach when fMLP was removed. Jasplakinolide did not inhibit changes in intracellular Ca(2+) seen after fMLP treatment, or inhibit neutrophil immobilisation induced by externally added Mn(2+). Thus cytoskeletal rearrangement was directly implicated in upregulation and, later, downregulation of CD11b/CD18 binding. Inhibition of RhoA with C3-transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulation; however, neither the conversion of rolling to stationary adhesion, nor the ability of neutrophils to detach on removal of the stimulus, were inhibited. Thus, Rho may regulate actin polymerisation and motility in neutrophils, but did not appear to control integrin-mediated adhesion itself. Integrin binding may be promoted by disruption of links to the cytoskeleton, effected through depolymerisation of actin or cleavage of linking protein talin by calpain. Disruption of actin filaments with cytochalasin D did not, however, cause integrin-mediated immobilisation of rolling neutrophils. Although the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP, this was only at doses where actin polymerisation was also ablated. We suggest that the cytoskeleton actively regulates binding conformation of CD11b/CD18 as well as its mobility in the membrane.

Bombesin-evoked gastrin release and calcium signaling in human antral G cells in culture

1999 ◽  
Vol 276 (1) ◽  
pp. G227-G237 ◽  
Author(s):  
Paul E. Squires ◽  
R. Mark Meloche ◽  
Alison M. J. Buchan
Keyword(s):  
Cell Culture ◽  
Calcium Signaling ◽  
Gastrin Release ◽  
Voltage Gated ◽  
Gastrin Releasing Peptide ◽  
G Cells ◽  
Peptide Family ◽  
Inositol 1,4,5 Trisphosphate ◽  
Voltage Dependent ◽  
Intracellular Ca

Amplification of mRNA from a human antral cell culture preparation demonstrated the presence of two receptors of the bombesin and gastrin-releasing peptide family, GRPR-1 and BRS-3. Single cell microfluorometry demonstrated that most cells that exhibited bombesin-evoked changes in intracellular Ca2+ concentration were gastrin immunoreactive, indicating that antral G cells express the GRPR subtype. There were two components to the intracellular Ca2+ response: an initial nitrendipine-insensitive mobilization followed by a sustained phase that was inhibited by removal of extracellular Ca2+ and 20 mM caffeine and was partially inhibited by 10 μM nitrendipine. Preexposure of cells to thapsigargin and caffeine prevented the response to bombesin, indicating activation of inositol 1,4,5-trisphosphate (IP3)-sensitive stores. Gastrin release could be partially reversed by removal of extracellular Ca2+ and blockade of L-type voltage-dependent Ca2+ channels, indicating that a component of the secretory response to bombesin was dependent on Ca2+ influx. These data demonstrated that bombesin-stimulated gastrin release from human antral G cells resulted from activation of GRPRs and involved both release of intracellular Ca2+ and influx of extracellular Ca2+through a combination of L-type voltage-gated and IP3-gated Ca2+ channels.

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